This application is a 371 of PCT/GB00/04875 filed Dec. 18, 2000.
This invention relates to compounds that inhibit farnesylation of mutant ras gene products through inhibition of the enzyme farnesyl-protein transferase (FPTase). The invention also relates to methods of manufacturing the compounds, pharmaceutical compositions and methods of treating diseases, especially cancer, which are mediated through farnesylation.
Cancer is believed to involve alteration in expression or function of genes controlling cell growth and differentiation. Whilst not wishing to be bound by theoretical considerations the following text sets out the scientific background to ras in cancer. Ras genes are frequently mutated in tumours. Ras genes encode guanosine triphosphate (GTP) binding proteins which are believed to be involved in signal transduction, proliferation and malignant transformation. H-, K- and N-ras genes have been identified as mutant forms of ras (Barbacid M, Ann. Rev. Biochem. 1987, 56: 779-827). Post translational modification of ras protein is required for biological activity. Farnesylation of ras catalysed by FPTase is believed to be an essential step in ras processing. It occurs by transfer of the farnesyl group of farnesyl pyrophosphate (FPP) to a cysteine at the C-terminal tetrapeptide of ras in a structural motif called the CAAX box. After further post-translational modifications, including proteolytic cleavage at the cysteine residue of the CAAX box and methylation of the cysteine carboxyl, ras is able to attach to the cell membrane for relay of growth signals to the cell interior. In normal cells activated ras is believed to act in conjunction with growth factors to stimulate cell growth. In tumour cells it is believed that mutations in ras cause it to stimulate division even in the absence of growth factors (Travis J. Science 1993, 260: 1877-1878), possibly through being permanently in GTP activated form rather than cycled back to GDP inactivated form. Inhibition of farnesylation of mutant ras gene products will stop or reduce activation.
One class of known inhibitors of farnesyl transferase is based on farnesyl pyrophosphate analogues; see for example European patent application EP 534546 from Merck. Inhibitors of farnesyl transferase based on mimicry of the CAAX box have been reported. Reiss (1990) in Cell 62, 81-8 disclosed tetrapeptides such as CVIM (Cys-Val-Ile-Met). James (1993) in Science 260, 1937-1942 disclosed benzodiazepine based peptidomimetic compounds. Lerner (1995) in J. Biol. Chem. 270, 26802 and Eisai in International Patent Application WO 95/25086 disclosed further peptidomimetic compounds based on Cys as the first residue. EP 696593 and PCT/GB96/01810 disclose further farnesyl transferase inhibitors, including pyrrolidine derivatives.
The applicants have found that a particular substitution of the pyrrolidine provides particular advantages in terms of inhibition of farnesyl transferase.
According to one aspect of the present invention there is provided a compound of formula (I): 
wherein:
R1 and R2 are independently selected from H or a prodrug moiety;
R3 is hydrogen or halogen;
R4 is hydrogen or halogen;
L is xe2x80x94CHxe2x95x90CHxe2x80x94 or xe2x80x94CH2xe2x80x94Zxe2x80x94 where Z is NH or O;
Y is S, S(O) or S(O)2;
or a salt thereof, provided that at least one of R3 or R4 is other than hydrogen.
As used herein, the term xe2x80x9calkylxe2x80x9d refers to straight or branched chain groups, which may, unless otherwise stated have from 1 to 20 and preferably from 1 to 6 carbon atoms. The term xe2x80x9carylxe2x80x9d includes phenyl. The term xe2x80x9chaloxe2x80x9d includes fluoro, chloro, bromo and iodo.
The term xe2x80x9cheterocyclylxe2x80x9d or xe2x80x9cheterocyclicxe2x80x9d include groups having from 4 to 10 ring atoms, up to 5 of which are selected from oxygen, sulphur and nitrogen. The rings may be mono-, or bicyclic and each ring may be aromatic or non-aromatic in character. Nitrogen atoms may be substituted if the valency of the ring allows it, with either a hydrogen or substituent group, such as a alkyl substituent. Sulphur atoms in a heterocyclic ring may be oxidised to S(O) or S(O)2 groups.
Examples of aromatic 5- or 6-membered heterocyclic ring systems include imidazole, triazole, pyrazine, pyrimidine, pyridazine, pyridine, isoxazole, oxazole, isothiazole, thiazole and thiophene. A 9- or 10-membered bicyclic heteroaryl ring system is an aromatic bicyclic ring system comprising a 6-membered ring fused to either a 5 membered ring or another 6 membered ring. Examples of 5/6 and 6/6 bicyclic ring systems include benzofuran, benzimidazole, benzthiophene, benzthiazole, benzisothiazole, benzoxazole, benzisoxazole, pyridoimidazole, pyrimidoimidazole, quinoline, isoquinoline, quinoxaline, quinazoline, phthalazine, cinnoline and naphthyridine.
Preferably monocyclic heteroaryl rings contain up to 3 heteroatoms and bicyclic heteroaryl rings contain up to 5 heteroatoms. Preferred heteroatoms are N and S, especially N. In general, attachment of heterocyclic rings to other groups is via carbon atoms. Suitable heterocyclic groups containing only N as the heteroatom are pyrrole, pyridine, indole, quinoline, isoquinoline, imidazole, pyrazine, pyrimidine, purine and pteridine.
Hydrogenated or other substituted forms of the above aromatic rings, (which are not aromatic), such as tetrahydropyridyl rings are examples of non-aromatic heterocyclic groups.
Various forms of prodrugs are known in the art. For examples of such prodrug derivatives, see:
a) Design of Prodrugs, edited by H. Bundgaard, (Elsevier, 1985) and Methods in Enzymology, Vol. 42, p. 309-396, edited by K. Widder, et al. (Academic Press, 1985);
b) A Textbook of Drug Design and Development, edited by Krogsgaard-Larsen;
c) H. Bundgaard, Chapter 5 xe2x80x9cDesign and Application of Prodrugsxe2x80x9d, by H. Bundgaard p. 113-191 (1991);
d) H. Bundgaard, Advanced Drug Delivery Reviews, 8, 1-38 (1992);
e) H. Bundgaard, et al., Journal of Pharmaceutical Sciences, 77, 285 (1988); and
f) N. Kakeya, et al., Chem Pharm Bull, 32, 692 (1984).
Suitable examples of groups R1 are hydrogen or prodrug groups of formula R5C(O)xe2x80x94where R5 is an optionally substituted aryl or heterocyclyl group. In particular R5 is optionally substituted phenyl, optionally substituted pyridyl optionally substituted furyl, optionally substituted isoxazole, optionally substituted tetrahydropyridyl or optionally substituted tetrahydrofuryl.
Suitable substituents for R5 include alkyl groups such as methyl, haloalkyl groups such as trifluoromethyl, hydroxy, alkoxy such as methoxy or cyano.
Preferably R5 is phenyl, pyridyl or N-methyl-tetrahydropyridyl.
Examples of prodrugs groups for R2 are in vivo cleavable ester groups of a pharmaceutically-acceptable ester which is cleaved in the human or animal body to produce the parent acid. Suitably R2 together with the carboxy group to which it is attached forms a pharmaceutically-acceptable esters such as C1-6alkyl esters or C1-6cycloalkyl esters, for example methyl, ethyl, propyl, iso-propyl, n-butyl or cyclopentyl; C1-6alkoxymethyl esters, for example methoxymethyl; C1-6alkanoyloxymethyl esters, for example pivaloyloxymethyl; phthalidyl esters; C3-8cycloalkoxycarbonyloxyC1-6alkyl esters, for example 1-cyclohexylcarbonyloxyethyl; 1,3-dioxolan-2-ylmethyl esters, for example 5-methyl-1,3-dioxolan-2-ylmethyl; C1-6alkoxycarbonyloxyethyl esters, for example 1-methoxycarbonyloxyethyl; aminocarbonylmethyl esters and mono- or di-Nxe2x80x94(C1-6alkyl) versions thereof, for example N,N-dimethylaminocarbonylmethyl esters and N-ethylaminocarbonylmethyl esters, and pharmaceutically acceptable esters of optionally substituted heterocyclic groups.
Further examples of such prodrugs for R2 are in vivo cleavable amides of a compound of the invention. Suitably R2 together with the carboxy group to which it is attached forms a pharmaceutically-acceptable amide, preferably an Nxe2x80x94C1-6alkylamide and an N,N-di-(C1-6alkyl)amide, such as N-methyl, N-ethyl, N-propyl, N,N-dimethyl, N-ethyl-N-methyl or N,N-diethylamide.
Thus in particular, R2 is selected from hydrogen, a C1-4alkyl group such as isopropyl or cyclopentyl, or an optionally substituted heterocyclic group such as N-methyl-tetrahydropyridyl.
R3 is suitably a halo atom such as fluoro or chloro group, in particular fluorine.
R4 is preferably a hydrogen or fluorine, and in particular is hydrogen.
The linking group L is suitably a group of formula CH2xe2x80x94Zxe2x80x94 where Z is NH or O.
Preferably the linking group L is xe2x80x94CHxe2x95x90CHxe2x80x94. Both E and Z isomeric forms of such compounds form part of the invention together with mixtures thereof. In particular, compounds were geometrical isomerism is possible are preferably in E form.
Group Y is preferably a group S or S(O)2.
It is to be understood that, insofar as certain of the compounds of Formula I defined above may exist in optically active or racemic forms by virtue of one or more asymmetric carbon atoms, the invention includes in its definition any such optically active or racemic form which possesses the property of inhibiting FTPase. The synthesis of optically active forms may be carried out by standard techniques of organic chemistry well known in the art, for example by synthesis from optically active starting materials or by resolution of a racemic form. Similarly, inhibitory properties against FTPase may be evaluated using the standard laboratory techniques referred to hereinafter.
Chiral carbon atoms at the 2 and 4 positions of the pyrrolidine ring in Formula I are preferred in the (S) configuration.
The chiral carbon atom at the 2 position between the carbonyl and amine in Formula I is preferred in the (S) configuration.
Compounds of Formula I may form salts which are within the ambit of the invention. Pharmaceutically acceptable salts are preferred although other salts may be useful in, for example, isolating or purifying compounds.
When the compound contains a basic moiety it may form pharmaceutically acceptable salts with a variety of inorganic or organic acids, for example hydrochloric, hydrobromic, sulphuric, phosphoric, trifluoroacetic, citric or maleic acid. A suitable pharmaceutically-acceptable salt of the invention when the compound contains an acidic moiety is an alkali metal salt, for example a sodium or potassium salt, an alkaline earth metal salt, for example a calcium or magnesium salt, an ammonium salt or a salt with an organic base which affords a pharmaceutically-acceptable cation, for example a salt with methylamine, dimethylamine, trimethylamine, piperidine, morpholine or tris-(2-hydroxyethyl)amine. Particular salts of compounds of the invention are acetates, alkyl sulphonates such as methyl or ethyl sulphonate, fumarates, formates, succinates and gluconates.
Solvates, for example hydrates, are also within the ambit of the invention and may be prepared by generally known methods.
Particular examples of compounds of formula (I) are shown in Table 1
According to another aspect of the invention there is provided a compound of Formula I for use as a medicament.
Further according to the invention there is provided a compound of Formula I for use in preparation of a medicament for treatment of a disease mediated through farnesylation of ras, in particular cancer.
The compound is suitably formulated as a pharmaceutical composition for use in this way.
Thus, according to yet another aspect of the invention there is provided a pharmaceutical composition comprising a compound of formula I listed above together with a pharmaceutically acceptable diluent or carrier.
According to another aspect of the present invention there is provided a method of treating ras mediated diseases, especially cancer, by administering an effective amount of a compound of Formula I to a mammal in need of such treatment.
According to a further feature of the invention there is provided a compound of Formula I, or a pharmaceutically-acceptable salt thereof, for use in a method of treatment of the human or animal body by therapy.
The invention also provides the use of a compound of formula (I) in the preparation of a medicament for use in treating farnesylated ras mediated disease or medical condition such as cancers.
Specific cancers which may be treated by the compound or composition of the invention include:
carcinoma, including that of the bladder, breast, colon, kidney, liver, lung, ovary, pancreas, stomach, cervix, thyroid and skin;
hematopoietic tumors of lymphoid lineage, including acute lymphocytic leukemia, B-cell lymphoma and Burketts lymphoma;
hematopoietic tumors of myeloid lineage, including acute and chronic myelogenous leukemias and promyelocytic leukemia;
tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; and
other tumors, including melanoma, seminoma, tetratocarcinoma, neuroblastoma and glioma.
The compounds of Formula I are especially useful in treatment of tumors having a high incidence of ras mutation, such as colon, lung, and pancreatic tumors. By the administration of a composition having one (or a combination) of the compounds of this invention, development of tumors in a mammalian host is reduced.
Compounds of Formula I may also be useful in the treatment of diseases other than cancer that may be associated with signal transduction pathways operating through Ras, e.g., neuro-fibromatosis.
Compounds of Formula I may also be useful in the treatment of diseases associated with CAAX-containing proteins other than Ras (e.g., nuclear lamins and transducin) that are also post-translationally modified by the enzyme farnesyl protein transferase.
The compositions of the invention may be in a form suitable for oral use (for example as tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs), for topical use (for example as creams, ointments, gels, or aqueous or oily solutions or suspensions), for administration by inhalation (for example as a finely divided powder or a liquid aerosol), for administration by insufflation (for example as a finely divided powder) or for parenteral administration (for example as a sterile aqueous or oily solution for intravenous, subcutaneous, intramuscular or intramuscular dosing or as a suppository for rectal dosing).
The compositions of the invention may be obtained by conventional procedures using conventional pharmaceutical excipients, well known in the art. Thus, compositions intended for oral use may contain, for example, one or more colouring, sweetening, flavouring and/or preservative agents.
Suitable pharmaceutically acceptable excipients for a tablet formulation include, for example, inert diluents such as lactose, sodium carbonate, calcium phosphate or calcium carbonate, granulating and disintegrating agents such as corn starch or algenic acid; binding agents such as starch; lubricating agents such as magnesium stearate, stearic acid or talc; preservative agents such as ethyl or propyl p-hydroxybenzoate, and anti-oxidants, such as ascorbic acid. Tablet formulations may be uncoated or coated either to modify their disintegration and the subsequent absorption of the active ingredient within the gastrointestinal tract, or to improve their stability and/or appearance, in either case, using conventional coating agents and procedures well known in the art.
Compositions for oral use may be in the form of hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules in which the active ingredient is mixed with water or an oil such as peanut oil, liquid paraffin, or olive oil.
Aqueous suspensions generally contain the active ingredient in finely powdered form together with one or more suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents such as lecithin or condensation products of an alkylene oxide with fatty acids (for example polyoxethylene stearate), or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives (such as ethyl or propyl p-hydroxybenzoate; anti-oxidants (such as ascorbic acid), colouring agents, flavouring agents, and/or sweetening agents (such as sucrose, saccharine or aspartame).
Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil (such as arachis oil, olive oil, sesame oil or coconut oil) or in a mineral oil (such as liquid paraffin). The oily suspensions may also contain a thickening agent such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set out above, and flavouring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.
Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water generally contain the active ingredient together with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients such as sweetening, flavouring and colouring agents, may also be present.
The pharmaceutical compositions of the invention may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, such as olive oil or arachis oil, or a mineral oil, such as for example liquid paraffin or a mixture of any of these. Suitable emulsifying agents may be, for example, naturally-occurring gums such as gum acacia or gum tragacanth, naturally-occurring phosphatides such as soya bean, lecithin, an esters or partial esters derived from fatty acids and hexitol anhydrides (for example sorbitan monooleate) and condensation products of the said partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening, flavouring and preservative agents.
Syrups and elixirs may be formulated with sweetening agents such as glycerol, propylene glycol, sorbitol, aspartame or sucrose, and may also contain a demulcent, preservative, flavouring and/or colouring agent.
The pharmaceutical compositions may also be in the form of a sterile injectable aqueous or oily suspension, which may be formulated according to known procedures using one or more of the appropriate dispersing or wetting agents and suspending agents, which have been mentioned above. A sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example a solution in 1,3-butanediol.
Suppository formulations may be prepared by mixing the active ingredient with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Suitable excipients include, for example, cocoa butter and polyethylene glycols.
Topical formulations, such as creams, ointments, gels and aqueous or oily solutions or suspensions, may generally be obtained by formulating an active ingredient with a conventional, topically acceptable, vehicle or diluent using conventional procedure well known in the art.
Compositions for administration by insufflation may be in the form of a finely divided powder containing particles of average diameter of, for example, 30xcexc or much less, the powder itself comprising either active ingredient alone or diluted with one or more physiologically acceptable carriers such as lactose. The powder for insufflation is then conveniently retained in a capsule containing, for example, 1 to 50 mg of active ingredient for use with a turbo-inhaler device, such as is used for insufflation of the known agent sodium cromoglycate.
Compositions for administration by inhalation may be in the form of a conventional pressurised aerosol arranged to dispense the active ingredient either as an aerosol containing finely divided solid or liquid droplets. Conventional aerosol propellants such as volatile fluorinated hydrocarbons or hydrocarbons may be used and the aerosol device is conveniently arranged to dispense a metered quantity of active ingredient.
For further information on Formulation the reader is referred to Chapter 25.2 in Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon Press 1990.
The amount of active ingredient that is combined with one or more excipients to produce a single dosage form will necessarily vary depending upon the host treated and the particular route of administration. For example, a formulation intended for oral administration to humans will generally contain, for example, from 0.5 mg to 2 g of active agent compounded with an appropriate and convenient amount of excipients which may vary from about 5 to about 98 percent by weight of the total composition. Dosage unit forms will generally contain about 1 mg to about 500 mg of an active ingredient. For further information on Routes of Administration and Dosage Regimes the reader is referred to Chapter 25.3 in Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon Press 1990.
The size of the dose for therapeutic or prophylactic purposes of a compound of the Formula I will naturally vary according to the nature and severity of the conditions, the age and sex of the animal or patient and the route of administration, according to well known principles of medicine. As mentioned above, compounds of the Formula I are useful in treating diseases or medical conditions which are due alone or in part to the effects of farnesylation of ras.
In using a compound of the Formula I for therapeutic or prophylactic purposes it will generally be administered so that a daily dose in the range, for example, 0.5 mg to 75 mg per kg body weight is received, given if required in divided doses. In general lower doses will be administered when a parenteral route is employed. Thus, for example, for intravenous administration, a dose in the range, for example, 0.5 mg to 30 mg per kg body weight will generally be used. Similarly, for administration by inhalation, a dose in the range, for example, 0.5 mg to 25 mg per kg body weight will be used. Oral administration is however preferred.
Compounds of this invention may be useful in combination with known anti-cancer and cytotoxic agents. If formulated as a fixed dose such combination products employ the compounds of this invention within the dosage range described herein and the other pharmaceutically active agent within its approved dosage range. Sequential use is contemplated when a combination formulation is inappropriate.
Although the compounds of the Formula I are primarily of value as therapeutic agents for use in warm-blooded animals (including man), they are also useful whenever it is required to inhibit the effects of activation of ras by farnesylation. Thus, they are useful as pharmacological standards for use in the development of new biological tests and in the search for new pharmacological agents.
According to another aspect of the present invention there is provided individual compounds produced as end products in the Examples set out below and salts thereof.
A compound of the invention, or a salt thereof, may be prepared by any process known to be applicable to the preparation of such-compounds or structurally related compounds. Such processes are illustrated by the following representative schemes in which variable groups have any of the meanings defined for Formula I unless stated otherwise. Functional groups may be protected and deprotected using conventional methods. For examples of protecting groups such as amino and carboxylic acid protecting groups (as well as means of formation and eventual deprotection), see T. W. Greene and P. G. M. Wuts, xe2x80x9cProtective Groups in Organic Synthesisxe2x80x9d, Second Edition, John Wiley and Sons, New York, 1991. Note abbreviations used have been listed immediately before the Examples below.
Protecting groups may be removed by any convenient method as described in the literature or known to the skilled chemist as appropriate for the removal of the protecting group in question, such methods being chosen so as to effect removal of the protecting group with minimum disturbance of groups elsewhere in the molecule.
Specific examples of protecting groups are given below for the sake of convenience, in which xe2x80x9clowerxe2x80x9d signifies that the group to which it is applied preferably has 1-4 carbon atoms. It will be understood that these examples are not exhaustive. Where specific examples of methods for the removal of protecting groups are given below these are similarly not exhaustive. The use of protecting groups and methods of deprotection not specifically mentioned is of course within the scope of the invention.
A carboxy protecting group may be the residue of an ester-forming aliphatic or araliphatic alcohol or of an ester-forming silanol (the said alcohol or silanol preferably containing 1-20 carbon atoms).
Examples of carboxy protecting groups include straight or branched chain (1-12C)alkyl groups (e.g. isopropyl, t-butyl); lower alkoxy lower alkyl groups (e.g. methoxymethyl, ethoxymethyl, isobutoxymethyl; lower aliphatic acyloxy lower alkyl groups, (e.g. acetoxymethyl, propionyloxymethyl, butyryloxymethyl, pivaloyloxymethyl); lower alkoxycarbonyloxy lower alkyl groups (e.g. 1-methoxycarbonyloxyethyl, 1-ethoxycarbonyloxyethyl); aryl lower alkyl groups (e.g. p-methoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, benzhydryl and phthalidyl); tri(lower alkyl)silyl groups (e.g. trimethylsilyl and t-butyldimethylsilyl); tri(lower alkyl)silyl lower alkyl groups (e.g. trimethylsilylethyl); and (2-6C)alkenyl groups (e.g. allyl and vinylethyl).
Methods particularly appropriate for the removal of carboxyl protecting groups include for example acid-, metal- or enzymically-catalysed hydrolysis.
Examples of hydroxy protecting groups include lower alkenyl groups (e.g. allyl); lower alkanoyl groups (e.g. acetyl); lower alkoxycarbonyl groups (e.g. t-butoxycarbonyl); lower alkenyloxycarbonyl groups (e.g. allyloxycarbonyl); aryl lower alkoxycarbonyl groups (e.g. benzoyloxycarbonyl, p-methoxybenzyloxycarbonyl, o-nitrobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl); tri lower alkyl/arylsilyl groups (e.g. trimethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl); aryl lower alkyl groups (e.g. benzyl) groups; and triaryl lower alkyl groups (e.g. triphenylmethyl).
Examples of amino protecting groups include formyl, aralkyl groups (e.g. benzyl and substituted benzyl, e.g. p-methoxybenzyl, nitrobenzyl and 2,4-dimethoxybenzyl, and triphenylmethyl); di-p-anisylmethyl and furylmethyl groups; lower alkoxycarbonyl (e.g. t-butoxycarbonyl); lower alkenyloxycarbonyl (e.g. allyloxycarbonyl); aryl lower alkoxycarbonyl groups (e.g. benzyloxycarbonyl, p-methoxybenzyloxycarbonyl, o-nitrobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl; trialkylsilyl (e.g. trimethylsilyl and t-butyldimethylsilyl); alkylidene (e.g. methylidene); benzylidene and substituted benzylidene groups.
Methods appropriate for removal of hydroxy and amino protecting groups include, for example, acid-, base, metal- or enzymically-catalysed hydrolysis, or photolytically for groups such as o-nitrobenzyloxycarbonyl, or with fluoride ions for silyl groups.
Examples of protecting groups for amide groups include aralkoxymethyl (e.g. benzyloxymethyl and substituted benzyloxymethyl); alkoxymethyl (e.g. methoxymethyl and trimethylsilylethoxymethyl); tri alkyl/arylsilyl (e.g. trimethylsilyl, t-butyldimethylsily, t-butyldiphenylsilyl); tri alkyl/arylsilyloxymethyl (e.g. t-butyldimethylsilyloxymethyl, t-butyldiphenylsilyloxymethyl); 4-alkoxyphenyl (e.g. 4-methoxyphenyl); 2,4-di(alkoxy)phenyl (e.g. 2,4-dimethoxyphenyl); 4-alkoxybenzyl (e.g. 4-methoxybenzyl); 2,4-di(alkoxy)benzyl (e.g. 2,4-di(methoxy)benzyl); and alk-1-enyl (e.g. allyl, but-1-enyl and substituted vinyl e.g. 2-phenylvinyl).
Aralkoxymethyl, groups may be introduced onto the amide group by reacting the latter group with the appropriate aralkoxymethyl chloride, and removed by catalytic hydrogenation. Alkoxymethyl, tri alkyl/arylsilyl and tri alkyl/silyloxymethyl groups may be introduced by reacting the amide with the appropriate chloride and removing with acid; or in the case of the silyl containing groups, fluoride ions. The alkoxyphenyl and alkoxybenzyl groups are conveniently introduced by arylation or alkylation with an appropriate halide and removed by oxidation with ceric ammonium nitrate. Finally alk-1-enyl groups may be introduced by reacting the amide with the appropriate aldehyde and removed with acid.
The invention also provides a process for preparing a compound of formula (I) as defined above which process The invention also provides a process for preparing a compound of formula (I) as defined above which process comprises reacting a compound of formula (II): 
where L, R3 and R4 are as defined in relation to formula (I), R1xe2x80x2 is a group R1 as defined in relation to formula (I) or a precursor thereof, and R5 is a protecting group such as BOC or ALLOC with a compound of formula (III): 
where Y is as defined in relation to formula (I) and R2xe2x80x2 is a group R2 as defined in relation to formula (I) or a precursor thereof;
and thereafter if desired or necessary, carrying out one or more of the following steps:
a) removing protecting groups R5;
a) converting any precursor groups R1xe2x80x2 and R2xe2x80x2 to groups R1 and R2; and
b) changing said groups to different R1, R2 groups.
The reaction between compounds of formula (II) and (III) is suitably effected in an organic solvent such as dichloromethane in the presence of a base such as DMAP and EDC. Moderate temperatures for example of from 0 to 50xc2x0 C., conveniently ambient temperature, are employed.
Precursor groups R1xe2x80x2 and R2xe2x80x2 may include protecting groups such as esters, which are not pharmaceutically acceptable. These may be converted to hydrogen or other prodrug groups using conventional methods as illustrated below.
Removal of protecting groups R5 can be carried out using conventional methods such as reaction with TFA and/or triethylsilane.
Compounds of formula (II) are suitably prepared by deprotecting a compound of formula (IV): 
where R1xe2x80x2, R3, R4, R5 and L are as defined in relation to formula (II) and R6 is a protecting group, in particular an alkyl group such as methyl. Deprotection is suitably effected using a strong base such as an alkali metal hydroxide, in particular sodium hydroxide. The reaction is suitably effected in a solvent such as aqueous alcohol and in particular aqueous methanol, at elevated temperatures, conveniently at the reflux temperature of the solvent.
Compounds of formula (IV) where L is xe2x80x94CH2NHxe2x80x94 may be prepared by coupling a compound of formula (V): 
where R3, R4 and R6 are as defined above; with an aldehyde of formula (VI): 
where R1xe2x80x2 and R5 are as defined above.
Suitable coupling conditions include the use of a reducing agent (e.g. NaCNBH3, BH3, hydrogen plus catalyst, LiHBEt3, di-isobutyl-aluminiumhydride, lithium aluminium hydride, sodium borohydride) in the presence of a suitable solvent e.g.methanol or ethanol and acetic acid.
Aldehydes of formula (VI) may be prepared by reduction of the compounds of formula (VII): 
where R1xe2x80x2 and R5 are as defined above and R6 is alkyl such as methyl and R7 is alkoxy such as methoxy.
Suitably powerful reducing agents such as lithium aluminium hydride are employed. The reaction is carried out in a solvent such as tetrahydrofuran at low temperatures, for example from xe2x88x9250 to 0xc2x0 C., in particular at about xe2x88x9220xc2x0 C.
Compounds of formula (VII) are suitably prepared by reacting a compound of formula (VIII): 
where R4 is a leaving group such a methansulphonyloxy group, which a compound of formula (IX): 
where R1xe2x80x2 is as defined above and in particular is a triphenylmethyl or trityl group. Reaction conditions would be apparent to the skilled person, but in general, the reaction is effected in an organic solvent such as dimethylformamide (DMF) at moderate temperatures, for example of from 0 to 60xc2x0 C. and preferably at about 40xc2x0 C.
Compounds of formula (VIII) may be prepared by reacting compounds of formula (X): 
where R5, R6 and R7 are as defined above, with a compound of formula (XI)
R8xe2x80x94Zxe2x80x83xe2x80x83(XI)
where R8 is as defined above and Z is a leaving group such as halogen, in particular chlorine. The reaction is suitably effected in an organic solvent such as dichloromethane in the presence of a weak base such as triethylamine. Moderate to low temperatures, for example, from xe2x88x9210 to 0xc2x0 C. are suitably employed.
Compounds of formula (X) are suitably prepared by reacting a compound of formula (XII): 
where R5 is as defined above, with a compound of formula (XIII): 
where R6 and R7 are as defined above. A particular example of a compound of formula (XII) is N,O-dimethylhydroxylamine. The reaction is suitably effected in the presence of a base (such as DCCI and DMAP) and in an organic solvent such as dichloromethane.
Compounds of formula (XII) may be prepared by N-protection of the corresponding hydroxy proline derivative using known methods.
Compounds of formula (V) are suitably prepared by hydrogenation of a compound of formula (XIV): 
where R3, R4 and R6 are as defined above. Hydrogenation is suitably effected in the presence of a catalyst such as a palladium catalyst.
Compounds of formula (XIV) are suitably prepared by reacting a compound of formula (XV): 
where R6 is as defined above and Zxe2x80x2 is a leaving group such as halogen and in particular bromine, with a compound of formula (XVI): 
where R3 and R4 are as defined above. The reaction is suitably effected in the presence of a reagent such as ceasium fluoride, and a catalyst such as a palladium catalyst (e.g. tetrakis(triphenylphsophine) palladium(0). A suitable solvent for the reaction is dimethoxyethane and the reaction can be effected under reflux conditions.
Compounds of formula (IV) where L represents xe2x80x94CH2Oxe2x80x94 may be prepared by reacting a compound of formula (XVII): 
where R3, R4 and R6 are as defined above,
with a compound of formula (XVIII): 
where R1xe2x80x2 and R5 are as defined above. The reaction is suitably effected under conditions similar to those described above for the reaction between compounds of formulae (V) and (VI).
Compounds of formula (XVIII) are suitably prepared by reduction of a compound of formula (VI), for example using a reducing agent such as lithium aluminium hydride. Reduction is carried out under conventional conditions in a solvent such as tetrahydrofuran.
Compounds of formula (XVII) may be prepared by protection of the corresponding carboxylic acid, for example by esterifying the acid using an alcohol, in particular an alkyl alcohol such as methanol. The reaction is suitably effected in the presence of sulphuryl chloride or the like, at elevated temperatures, conveniently at the reflux temperature of the solvent.
The acid itself may be prepared by deprotection of a compound of formula (XIX): 
where R3 and R4 are as defined above and R10 and R11 are protecting groups such as alkyl, and in particular methyl groups. Suitable deprotection conditions include heating the compound with a suitable reagent such as pyridine hydrochloride to high temperatures such, for example from 200 to 250xc2x0 C., and preferably at about 220xc2x0 C.
Compounds of formula (XIX) are obtained by reaction of a compound of formula (XX): 
where R10 and R11 are as described above and Zxe2x80x3 is a leaving group such as halogen, in particular bromine, with a compound of formula (XVI) as defined above, using conditions similar to those described for the reaction of a compound of formula (XV) with a compound of formula (XVI).
Compounds of formula (IV) where L is xe2x80x94CHxe2x95x90CHxe2x80x94 are suitably prepared by reacting a compound of formula (VI) as defined above with a compound of formula (XXI): 
where R3, R4 and R6 are as defined above and R12 is a phosphate ion such as diethylphosphate, or a triphenylphosphine group. The reaction is a Wittig reaction and is suitably carried out under conventional conditions. Suitable reaction conditions include the use of a base (e.g. potassium carbonate, metal hydride, metal alkoxide) in the presence of an organic solvent (e.g. THF, toluene, DMSO) optionally in the presence of an aqueous solvent (2-phase system) and optionally in the presence of a catalyst complexing agent which solubilises alkali metal ions in non-polar solvents such as 1,4,7,10,13-pentaoxacyclopentadecane (also called 15-Crown-5) or 1,4,7,10,13,16-hexaoxacyclooctadecane (also called 18-Crown-6).
Compounds of formula (XXI) are suitably obtained by reacting a compound of formula (XXII): 
where R3, R4 and R6 are as defined above and Zxe2x80x2xe2x80x3 is a leaving group such as halogen, and in particular bromine, with a phosphite such as triethyl phosphite. Reflux conditions are suitably employed and an inert atmosphere may be provided.
Compounds of formula (XXI) may be produced using methods described for example in PCT/GB98/00230. Preparation details are summarised further in Scheme 2 hereinafter.
If necessary or required, groups R1 and R2 may be changed for different such groups after any of the above preparation methods using conventional chemistry and examples of this are provided hereinafter.
Biological activity was tested as follows. Farnesyl protein transferase (FPT) was partially purified from human placenta by ammonium sulphate fractionation followed by a single Q-Sepharose(copyright) (Pharmacia, Inc) anion exchange chromatography essentially as described by Ray and Lopez-Belmonte (Ray K P and Lopez-Belmonte J (1992) Biochemical Society Transations 20 494-497). The substrate for FPT was Kras (CVIM C-terminal sequence). The cDNA for oncogenic val12 variant of human c-Ki-ras-2 4B was obtained from the plasmid pSW11-1 (ATCC). This was then subcloned into the polylinker of a suitable expression vector e.g. pIC147. The Kras was obtained after expression in the E. coli strain, BL21. The expression and purification of c-KI-ras-2 4B and the val12 variant in E. coli has also been reported by Lowe et al (Lowe P N et al. J. Biol. Chem. (1991) 266 1672-1678).
Incubations with enzyme contained 300 nM tritiated farnesyl pyrophosphate (DuPont/New England Nuclear), 120 nM ras-CVIM, 50 mM Tris HCl pH 8.0, 5 mM MgCl2, 10 xcexcM ZnCl2, 5 mM dithiotheitol and compounds were added at appropriate concentrations in DMSO (3% final concentration in test and vehicle control). Incubations were for 20 minutes at 37xc2x0 and were stopped with acid ethanol as described by Pompliano et al. (Pompliano D L et al (1992) 31 3800-3807). Precipitated protein was then collected onto glass fibre filter mats (B) using a Tomtec(copyright) cell harvester and tritiated label was measured in a Wallac(copyright) 1204 Betaplate scintillation counter.
Although the pharmacological properties of the compounds of the Formula I vary with structural change as expected, in general compounds of the Formula I possess an IC50 in the above test in the range, for example, 0.01 to 200 xcexcM.
The invention will now be illustrated in the following non-limiting Examples in which, unless otherwise stated:
(i) evaporations were carried out by rotary evaporation in vacuo and work-up procedures were carried out after removal of residual solids by filtration;
(ii) operations were carried out at room temperature, that is in the range 18-25xc2x0 C. and under an atmosphere of an inert gas such as argon;
(iii) column chromatography (by the flash procedure) and medium pressure liquid chromatography (MPLC) were performed on Merck Kieselgel silica (Art. 9385) or Merck Lichroprep RP-18 (Art. 9303) reversed-phase silica obtained from E. Merck, Darmstadt, Germany;
(iv) yields are given for illustration only and are not necessarily the maximum attainable;
(v) the end-products of the Formula I have satisfactory microanalyses and their structures were confirmed by nuclear magnetic resonance (NMR) and mass spectral techniques; chemical shift values were measured on the delta scale; the following abbreviations have been used: s, singlet; d, doublet; t or tr, triplet; m, multiplet; br, broad;
(vi) intermediates were not generally fully characterised and purity was assessed by thin layer chromatographic, infra-red (IR) or NMR analysis;
(vii) melting points are uncorrected and were determined using a Mettler SP62 automatic melting point apparatus or an oil-bath apparatus; melting points for the end-products of the Formula I were determined after crystallisation from a conventional organic solvent such as ethanol, methanol, acetone, ether or hexane, alone or in admixture; and
(viii) the following abbreviations have been used: